Antitumor agent m-6672 and method of preparation



United States Patent This invention relates to a new composition of matter possessing antitumor properties and to a process for its production. In particular,'-the invention is concerned with the substance herein designated M-6672 and with a method for its production.

Although the two organisms appear to be similar in a number of respects, the following table lists the significant difierences and includes important characteristics which were not determined for S. rameus. Accordingly, the organism of the present invention is designated a new species and given the name Streptomyces terminospiralis indicating the spiral and coiled nature of the ends of its long spore chains. A living culture of S. terminospiralis has been deposited with the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, 111., and has the assigned number NRRL 3128.

DIFFERENTIAL CHARACTERISTICS OF S. TERMINOSPIRALIS AND S. RAMEUS Morphological S. terminospiralis S. ramms Sporophorcs Terminal coils, fists and spirals of three to five Incomplete spirals, loops or hooks.

turns on long spore chains. Length of spore chain. Commonly spores. Not reported. Branching of sporophores Mo no godial, singly lew pairs, and with terminal Monopodial, (further information locking).

0 us ers. Spore morphology Predominantly bacillar y or cylindrical with Oval to oblong.

length to diameter ratio 3:1. Spore surface (Electron microscopy) Smooth Not reported. Co or:

Sporulating aerial White to yellow White Grayish. Ditiusible pigment Czapeks Agan. None Yellow. Physiologic:

Melanin (tyrosine added to peptone-yeast extract iron agar).

Positive-heavy black pigment Possibly positive, brown pigment on nutrient agar, potato and gelatin.

H S production (Lead acetate strip test)..-" Positi e N ot reported. Haemolysis 0 Negative. Starch Hydrolysis Very weak. 2 mm. 22 days by iodine test. Weak to moderate. 8.0 to 18.0 mm. Proteolysis:

Gelatin, Plain 16 mm. 22 days Not reported. Gelatin, Nutrient d0 Doubtful. M111; N3 coagulation. Complete peptonization 14 Weak coagulation. Doubtlul peptonization.

ays. Carbohydrate Utilization:

Mannitol- Negative .1 Positive. Raffinos 11 Do. Salicin Negative. Xylose. 0. Temperature limit. Not reported. Antagonistic Properties: I

Antibacterial Activity Produces antibiotic similar to streptomycin by Produces streptomycin.

paper chromatography and antibacterial spectruJn. Antitumor Activity Positive N ot reported.

The novel substance M6672 is produced by cultivating on suitable nutrient media and under controlled conditions the hithertoundescribed microorganism Streptomyces terminosp iralis. This organism was isolated from a pasture soil near Dallas, Texas, USA. Its cultural, morphological, and physiological characteristics were compared with the published descriptions of known species and found to agree most closely with Streptomyces rameus, Okami et al., J. Antibiotics (Japan) 12A, 257262, 1959. The name S. galbus used in this publication was also used by Frommer in describing a new species of Streptomyces, Arch. Mikrobiologie 32, 195, 1959. The name Streptomyces rameus is applied to the organism of Okami et al. in the Actinomycetes, Volume 2, Classification, Identification and Descriptions of Genera and Species, Selm-an A. Waksman, 1961, The Wi-L liams and Wilkins Comp-any, Baltimore, Maryland.

Taxonomy of Streptomyces terminospiralis Except as otherwise indicated, growth characteristics were determined after incubation at 28 C. on mm. agar plates containing 20.0 ml. of the indicated media. Morphological characteristics by light microscopy were determined from both plates and micro slide cultures. Electron micrographs were obtained from agar plate growth. Determinations of color were: made by comparison with the color chips of The Color Harmony Manual, third edition, Jacobson, Granville and Foss, Container Corporation of America. Color names are those of the ISCC-NBS Method of Designating Colors and a Dictionary of Color Names, U.S. Department of Com merce, National Bureau of Standards, Circular 553,1955.

Agar plates were inoculated in such a way as to provide a band of confluent growth about 1.0 cm. wide on one side of the plate with more or less isolated colonies colonies were made from those usually not less than 1.0 over the remainder of the plate. Descriptions of isolated cm. from the nearest growth.

DESCRIPTIVE CHARACTERISTICS OF STREPTOMYC'ES TERMINOSPIRALIS Medium Day Growth Aerial Reverse Soluble pigment and Czapeks solution with 7 Good. Colonies 2.5-3.0 mm. Abundant, White Inner confluent 1% ea, None.

dextrose agar. heaped, central pit, light yellow; outer coneraeked. fluent and isolated colonies, 1V fb, mod. 7 yellow. 14 Isolated colonies 4.5 mm. Abundant aerial. 2 ga, light yellow both of Do.

crateriiorm with radial Abundant spores b above areas. folds, many with ruptured white. surface. 22 As day 14, 5.0 mm. with As day 14 Isolated colonies and edge Do.

slight extension below agar of confluent 2 kb, strong surface. yellow; Inner confluent 2 ga, light yellow.

Ca Malate Agar 7 Moderate. Colonies 3.0 mm. Sparse None. Moderate with feathery edges. digestion of malate in crowded areas. 14 Good. Isolated colonies flat Abundant aerial. None. Digestion 3.0

4.0 mm. with extension Abundant white mm. from edge of into agar. spores. confluent growth. 22 Essentially same as day 14- Spore color 2 ba, Uniformly 2 db, pale As day 14. Digestion yellowish white. yellow. 5.0 mm.

Glycerol Asparagine Agar. 7 Abundant. Colonies flat, Sparse 1 db, pale yellow green None.

4.0 mm. with erose edges. 14 2.0 mm. zone of clearing of Abundant aerial and 1% ea, light yellow Do,

medium around each spores b, white.

colony. Isolated colonies 6.0 mm. undulate edges.

22 Colonies 8.0 mm. with 4.0 As day 14 Center of confluent growth Do.

mm. low convex center, 2 ea, light yellow. Edge extension into agar 1.0 mm. and colonies 2 hb,

moderate yellow.

Inorganic salts soluble 7 Light to moderate. Pln- Very sparse starch agar. point colonies.

14 Moderate. Colonies 1.0 mm. Sparse aerial moderate 3 ga, light orange yellow. None. No hydrolysis with 1.0 mm. extension sporulation. by iodine test. into agar.

22 As day 14 Moderate aerial and 2 go, grayish yellow None. Hydrolysis abundant spores 2 ba, only under growth. yellowish white.

Yeast extract, soluble 7 Good. Colonies 3.5-5 mm. Sparse 2 fb, pale yellow None. Hydrolysis starch, dextrose agar. heaped centers with negative.

numerous radial folds.

14 Essentially same Sparse to moderate 2 hb, light to moderate None. Test for hydrolaerial and spores. yellow. ysis omitted.

22 Colonies 4.0 mm. convex Abundant aerial and 2 ga, light yellow None. 2.0 mm. zone with central papilla, spores 2 ba, yellowish of hydrolysis beyond entire edge. White. edge of confluent growth by iodine test.

Yeast-malt extract agar. 7 Abundant growth. Colonies Abundant aerial and 3 ne, strong yellowish Slight brownish. 4-5 nun. heaped with spores, white. brown. central pit and radial folds, undulate edge.

14 As above with colonies more b, white 2 mb, strong yellow None.

orateriform.

22 Colonies 10.0 mm. with 1-2 -do mm. extending into agar. Annular rings present near fiat outer edge of growth.

Tomato Paste, oatmeal 6 Abundant growth. Colonies Abundant aerial and agar adjusted pH 7.0 4-5 mm. with 0.5 mm. spores. b, white. with NaQH. No flat edge. Aerial most prolific growth with pH unat edge of growth. adjusted.

13 Colonies 5-7 mm. with Sameannular rings. 21 Same with extension of do growth into agar 1-2 mm.

Nutrient Agar 7 Fair to moderate. Rough Very scant 21c, moderate yellow Very slight brownish.

colonies 2.5 mm. slightly erose edges. 14 Colonies, irregular rough Very sparse 2 1c, strong yellow Very faint yellowish.

surface, flat 2-3 mm. 22 Moderate. Sparse aerial. do

Nutrient Gelatin (Tubes) 7 Abundant Abundant white aeriaL. Brownish black pig- 24 C. merit. Liquefaction 7.0 mm. 14 Growth sunk below surface Black pigment 10 mm.

to bottom of liquid portion. liquid. 22 Same Plain 12% Gelatin Duplicated nutrient gelatin-.. 16 mm. liquid same as nutrient gelatin.

Norm-Two other strcptomyces classified concurrently grew well on the unadjusted medium.

PHYSIOLOGICAL CHARACTERISTICS OF ASITREPTOMYGES TERMINOSPIRALIS Starch hydrolysis very weak. Haemolysis (human blood) positive. Proteolytic activity:

Gelatin (plain and nutrient)- moderate to good.

Same plus 1.0% L-tyrosine positive (In re intense pigment). H S From peptone-yeast-iron agar with filter paper strip containing lead acetate positive.

CARBOHYDRATE UTILIZATION BY STREPTOMYC'E'S TERMINOSPIRALIS Xylose Soluble starch Arabinose Salicin Rhamnose Glycerol Dextrose Mannitol Galactose Dulcitol Mannose Inositol Fructose Sorbitol Sorbose Na citrate Sucrose Na acetate Lactose Na-K tartrate Maltose Control (no carbon Cellobiose source) Ratfinose 1 Basal medium of Pridham and Gottlieb, J. Bact. 56 107', 1948. Determinations also made omitting C11S045H2O. Growth was more rapid and abundant in all cases of utilization with the modified medium.

Micromorphology of Streptomyces terminospiralis Microscopic examinations were made at intervals during growth of the organisms on a number of media including those of carbon utilization studies. In addition, slide cultures made with yeast extract malt extract agar were examined. The media were in general agreement in production of monopodi-ally branching sporophores from medium length aerial hyphae. Sporophores near the distal end of the hyphae arose closer together with some rebranching from secondary hyphae producing a clustered appearance. Sporophores sometimes occurred in pairs but verticils were absent. The sport chains were generally long containing up to as many as fifty spores and typically thirty or more. Some fully sporulated flexuous chains were seen as well as a few simple hooks and loops, but the majority produced fists, closed spirals and coils of three to five turns terminally.

Individual spores in a chain observed in slide cultures as well as on plate cultures were typically bacillary (cylindrical with rounded ends) with a smaller proportion of oval spores. Measurements were not taken but the length to diameter ratio approximated 3:1.

Electron micrographs confirmed the bacillary morphology and showed the spore surface to be of the smooth type.

Fermentation of Streptomyces terminospiralis NRRL 3128 is carried out in submerged form in an aqueous nutrient medium containing suitable sources of carbon and nitrogen and with appropriate agitation, aeration and temperature control. A temperature of 24-28 C. is

suitable for production of the antitumor agent in a period of 2-4 days. We prefer a 3 day fermentation at 24 C. The presence of the antitumor agent in the fermentation broth can be demonstrated by daily intraperitoneal injections of an appropriate dilution of the filtered broth into mice bearing the experimentally induced tumor Sarcoma 180 or Carcinoma 755. No microorganism has been found which is sensitive to antitumor agent M-6672 in concentrations suitable for assay. Since the animal test consumes at least 7 days, it is desirable to proceed with the isolation Without waiting for the assay. If desired, however, the fermented broth can be stored for several weeks at temperatures of 05 C.

The antitumor agent is isolated from the fermented broth by fractional precipitation with solvents. Antitumor agent M-6672 is virtually insoluble in such water miscible solvents as methanol and acetone and is very soluble in water. By evaporating most of the water from the filtered broth at reduced pressure, adding the solvent in portions and removing the precipitate after each addition of solvent, a series of precipitates is obtained which are shown by the standard Sarcoma 180 mouse test to contain the desired antitumor substance.

Antitumor agent M-6672 is a light brown powder very soluble in water, soluble in aqueous alcohol (methyl or ethyl) and insoluble in dry alcohols, acetone and other common organic solvents. It exhibits no optical rotation at the wave length of the sodium D-line and no characteristic absorption of ultraviolet light.

The following examples illustrate the formation, recovery and concentration of antitumor agent M-6672.

EXAMPLE l.-FERMENTATION IN SHAKEN FLASK Streptomyces terminospiralis was seeded from soil stock to an agar slant of the following composition and incubated for about five days at 28 C.

Glucose -g 4 Yeast extract g 4 Malt extract g 10 Agar g 20 H O liter 1 After incubation, the resulting growth was scraped from the agar slant and introduced into ml. of sterile medium of the following composition in a 500 m1. Erlenmeyer flask:

Soybean meal g 15 Glucose g 15 NaCl g 5 CaCO g 1 O liter 1 This flask was incubated for 48 hours at 28 C. on a rotary shaker describing a 2% inch eccentric at about 240 rpm. Following this, another flask containing sterile medium of the same composition was seeded with about 4% v./v. of the resultant growth from the first flask. This flask was also incubated for 48 hours at 28 C. with shaking.

For production, a 500 ml. Erlenmeyer flask containing 150 ml. of sterilized medium of the following composition was seeded with 4% v./v. of material from the second 48 hour flask. as before with the exception that the incubation period was for approximately 72 hours.

H O liter 1 Incubation and agitation for this flask was diluted with 12.9 liters of methanol.

Test No. Dilution for Dosage Tumor Percent hange Reduction 1 Undiluted 25/1. 45 83 do 25/. 98 75 2 0/8670 100 Test tumor diameter in centimeters/control tumor diameter in centimeters.

2 Test tumor weight in milligrams/control tumor weight in milligrams.

EXAMPLE 2.PRODUCTION IN SMALL FERMENTORS In each of 4 stainless steel fermentation tanks of 23 liters capacity is placed 12 liters of an aqueous nutrient medium having the following composition.

Per liter Glucose g 25 Soybean Meal g 20 Corn Steep Liquor g 5 NaCl g 5 C3003 g 1 Polypropylene Glycol ml 1 The pH is adjusted to 6.8 by addition of sodium carbonate and the tanks and their contents are sterilized by autoclaving at 121 C. for 60 minutes. After cooling, each tank is inoculated with 300 ml. of second passage seed culture prepared as in Example 1. The tanks and their contents are maintained at a temperature of 24 C. for 3 days during which time the contents are mechanically stirred and sterile air is forced into the bottom of each tank at a rate of 12 liters per minute.

At the end of the fermentation, the broth is filtered and concentrated by vacuum distillation to 2150 ml. and A precipitate is formed consisting of about half the solids originally present in the filtered broth. The precipitate is removed by filtration and discarded. To the filtrate is added 5 liters of acetone and the precipitate which forms at this point is recovered by filtration, dissolved in water and freezedried to yield the crude antitumor agent as a powder. Additional active precipitate is formed when another 5 liters of acetone is added to the filtrate. The solids remaining in solution after the second precipitation are inactive.

What I claim is:

1. A process for the production of M6672 which cornprises cultivating the organism Streptomyces terminospiralz's under submerged aerobic conditions in an aqueous culture medium containing assimilable sources of carbohydrates, organic nitrogen and inorganic salts until substantial antitumor activity is produced by said organism and fractionally precipitating with a water-miscible solvent M-6672 from said culture medium.

2. A process as claimed in claim 1 in which the organism employed is Streptomyces terminospiralis NRRL 3128.

3. A process as claimed in claim 2 in which the culture medium is maintained at a temperature of from 24-28 C. for a period of from 2 to 4 days.

4. A process as claimed in claim 2 which includes the steps of filtering and concentrating the culture medium and adding a water miscible solvent selected from the group consisting of methanol and acetone to fractionally precipitate the M6672.

5. A process as claimed in claim 4 in which the water miscible solvent employed is methanol.

6. The product M-6672 produced by the process of claim 1.

No references cited.

A. LOUIS M'ONACELL, Primary Examiner.

A. E. TANENHOLTZ, Assistant Examiner. 

1. A PROCESS FOR THE PRODUCTION OF M-6672 WHICH COMPRISES CULTIVATING THE ORGANISM STREPTOMYCES TERMINOSPIRRALIS UNDER SUBMERGED AEOBIC CONDITIONS IN AN AQUEOUS CULTURE MEDIUM CONTAINING ASSIMILABLE SOURCE OF CARBOHYDRATES, ORGANIC NITROGEN AND INORGANIC SALTS UNTIL SUBSTANITAL ATNITUMOR ACTIVITY IS PRODUCED BY SAID ORGANISM AND FRACTIONALLY PRECIPITATING WITH A WATER-MISCIBLE SOLVENT M-6672 FROM SAID CULTURE MEDIUM.
 3. A PROCESS AS CLAIMED IN CLAIM 2 IN WHICH THE CULTURE MEDIUM IS MAINTAINED AT A TEMPERATURE OF FROM 24*-28* C. FOR A PERIOD OF FROM 2 TO 4 DAYS. 